Many biotechnology companies have been interested in developing transgenic plants that would produce fructosyltransferase to facilitate the development of a sugar substitute that is sweet and not hydrolysed in the human stomach or small intestine, and having good organoleptic properties. A desirable substitute would not be degraded or absorbed in to the body, and would provide a much lower energy value than glucose, fructose, or sucrose.
Fructans are non-structural storage carbohydrates. This polymer consists mostly of repeating fructose units. Fructans occur in Monocots such as Poaceae, and Liliaceae, and in some Dicots, such as Compositae.
Fructans have not been commercially useful so far, due to the limited species they occur in and their low level of accumulation in those species. The function of a fructan is determined by the length of the fructan chain and the degree of polymerization (DP) of the monosaccharide. A DP value of 3 would mean that there are three monosaccharides (Gm Fn), where G-F is sucrose, G is glucose, and F is fructose. The glycosidic linkage which interconnects the fructose units can be a 2-1 or a 2-6 type, depending on the fructosyltransferase. The function of a fructan depends on its backbone, its DP, and the degree of branching.
Many fructan related patent applications disclose methods designed to produce low calorie sweeteners in plants that normally do not produce fructans. A number of applications report the production of transformed plants having differing degrees of polymerization and total amount of fructosyl and glycosyl residues.
The synthesis of fructans in bacteria is catalysed by one enzyme, levansucrase (Dedonder R). (1966) Levansucrase from Bacillus subtilus (Methods in Enzymology 8, 500-505). Bacterial sequences encoding FTF in S. mutans and levansucrase in B. subtilus are described by Sato and Kurimaitsu (1986). Isolation and characterisation of a fructosyltransferase gene from Streptococcus mutans GS-5 (1986). Bacterial genes transformed into host plants were able to facilitate the synthesis of fructans (Van der Meer et al. (1994)). Fructans have also been are described as a new carbohydrate sink in transgenic potato plants (Plant Cell 6, 561-570). An attempt to use the levansucrase gene for enhancing tomatoes was disclosed in WO89/12386, WO94/14970 and U.S. Pat. No. 6,147,280 (the teaching of U.S. Pat. No. 6,147,280 is hereby incorporated by reference) disclose use of the levansucrase encoded by the ftf gene of S. mutans, the levansucrase encoded by the SacB gene of B. subtilus, the 6-SFT gene from barley, and the FFT gene from Jerusalem artichoke (Helianthus tuberosus L) to produce oligosaccharides having 0 or 1 glucose residues and having 2-8 and preferably 2-3 fructose residues.
WO96/21023 describes transforming plant genes encoding 1-SST and 1-FFT individually into petunias and potatoes. This application describes 1-SST and 1-FFT as having essentially different enzymatic properties. 1-FFT was not able to catalyse the initial step of fructan synthesis, and 1-SST was not able to catalyse the formation of fructan polymers with a degree of polymerisation higher than 5. By employing 1-SST activity alone, it was only possible to synthesis oligofructans from sucrose having a degree of polymerization of up to 5. This application did not describe the synthesis of fructans with a higher degree of polymerization using sucrose as a substrate indicating that both 1-SST and 1-FFT are needed for higher degree of polymerization and that 1-FFT activity alone is insufficient to carry out the synthesis of fructans from sucrose.
There is a need for transgenic plants, prepared from plants that do not normally produce fructans, which accumulate higher DP fructans by the elongation of the isokestose produced by 1-SST and/or sucrose. There is also a need for the action of two enzymes which results in the formation of a mixture of fructan molecules with different chain lengths.